Modulating excitatory synaptic neurotransmission: potential treatment for neurological disease?

The origin and development of the spermatogenic cell lineage is reviewed, as well as spermatogonial kinetics in adult nonprimate mammals in relation to the cycle of the seminiferous epithelium, the emphasis being on spermatogonial stem cells. A hypothesis is presented for the transition from foetal germ cells, gonocytes, to adult type spermatogonia at the start of spermatogenesis. An overview is given of the present knowledge on the proliferation and differentiation of undifferentiated spermatogonia (spermatogonial stem cells and their direct descendants) and the regulation of these processes. It is concluded that the differentiation of the undifferentiated into differentiating type spermatogonia is a rather vulnerable moment during spermatogenesis and the models for studying this are described. Research into the molecular basis of the regulation of spermatogonial proliferation, differentiation and apoptosis is at its infancy and the first results are reviewed. An exciting new research tool is the spermatogonial stem cell transplantation technique which is described. Finally, reviewing the nature of human germ cell tumours it is concluded that at present there are no animal or in vitro models to study these tumours experimentally.     

Polyamines and regulation of spermatogenesis: selective stimulation of late spermatogonia in transgenic mice overexpressing the human ornithine decarboxylase gene

Polyamines are believed to participate in the induction of cell growth, differentiation, and proliferation, but their role in spermatogenesis has remained obscure. Two transgenic mouse lines (K2 and K15) that overexpress the human ornithine decarboxylase (ODC) gene coding for a rate-controlling enzyme in polyamine biosynthesis and, hence, contain high levels of tissue putrescine have been used to study the stage-specific role of ODC in spermatogenesis. In K2 mice with 30-fold testicular ODC overexpression, [3H]thymidine incorporation at stages I-VI of the cycle of the seminiferous epithelium was significantly above the control level. This may reflect a specific stimulation of DNA synthesis in type A4, intermediate, and type B spermatogonia. The K15 mice that have about 70-fold ODC overexpression showed an elevation of DNA synthesis only at stage V of the cycle, suggesting a specific dependence of type B spermatogonia on putrescine. In K15 mice, [3H]thymidine incorporation of stage VIII tubule segments was decreased, suggesting that excess amounts of putrescine selectively inhibit meiotic DNA synthesis. We propose that putrescine has strictly selective local stimulatory and inhibitory actions during spermatogenic DNA synthesis, and that its excess amounts ultimately may lead to decreased fertility.     

Motor function of the proximal stomach and visceral perception in gastro-oesophageal reflux disease

Oxytocin (OT) is present in the mammalian testis and has been shown to play a role in the modulation of seminiferous tubule contractility and steroidogenesis. However, stage-specific effects of the peptide have not been previously investigated. In this study, computer-assisted analysis and time-lapse videomicrography were used to investigate basal contractility and the response to OT of seminiferous tubules at specific stages of the spermatogenic cycle. Adult rat testes were placed in fresh oxygenated DMEM F12 medium, decapsulated, and the tubules gently teased apart. Stages were identified by transillumination and a 10 mm section of tubule at each of stages IV-V, VII-VIII and XIII-I was placed in a microslide chamber and perifused with medium. After a control period of 3 h, OT (2 nM) was given for 1 h, followed by another control period of 1 h. The experiment was repeated using tubules from different rats and data were analysed to give arbitrary units of tubule contractility. Contractility was observed in all the tubules studied and the contractile activity was shown to vary depending on the stage of the spermatogenic cycle. Mean basal contractility at stages VII-VIII, the time when sperm are shed from the epithelium, was significantly lower than that at stages IV-V and XIII-I. The response of the tubules to OT was also stage-dependent, with the peptide producing the largest increases in contractile activity at stages VII-VIII and having no effect at stages IV-V. We postulate that these stage-specific differences in basal and OT-stimulated contractility may be important in co-ordinating the movement of developing germ cells towards the lumen of the seminiferous epithelium and in the process of spermiation.     

Stage-specific expression of the Kit receptor and its ligand (KL) during male gametogenesis in the mouse: a Kit-KL interaction critical for meiosis

The Kit receptor and its ligand KL, which together constitute an essential effector at various stages of embryonic development, are both present during adult gametogenesis. In the testis, KL is expressed in Sertoli cells, and Kit in germ cells, starting at the premeiotic stages. A series of observations indicated previously a role in spermatogonia survival, without excluding a possible function at later stages. We identified a complex pattern of expression of the two components in the adult murine testis, suggestive of a role in the meiotic progression of spermatocytes. At stages VII-VIII of the cycle of the seminiferous epithelium, the time when spermatocytes enter meiosis, the membrane-associated form of KL extends on the Sertoli cell from the peripheral to the adluminal compartment of the tubule. We also found that the receptor is present on the surface of germ cells up to the pachytene stage. The availability of differentiated Sertoli cell lines, which express the KL protein and support part of the maturation of germ cells in coculture, allowed us to ask whether, in the in vitro reconstructed system, transit of spermatocytes through meiosis requires the Kit-KL interaction. Addition of a blocking monoclonal antibody against the Kit receptor (ACK2) inhibited extensively the appearance of haploid cells and the expression of a haploid-phase-specific gene (Prm1). Recognition of the supporting Sertoli cell by germ cells was not affected, indicating a requirement for the activity of the receptor for either entering or completing meiosis. Involvement of the membrane-associated form of the ligand was suggested by the observation that addition of the soluble form of KL was equally inhibitory.     

Duration of spermatogenesis and sperm transit time through the epididymis in the Piau boar

The Piau boar is a rustic breed of economical importance in Brazil. The duration of spermatogenesis and sperm transit through the epididymis in Piau boars was estimated using intratesticular injections of tritiated thymidine. Animals were sacrificed 1 h, 7 days, 14 days, 21 days, 34 days, and 36 days after injections. Each cycle of spermatogenesis in Piau boars lasts 9 +/- 0.2 days. At least 9 days are necessary for spermatozoa to traverse the entire epididymis. Considering that the total duration of spermatogenesis takes about 4.5 seminiferous epithelium cycles, spermatogenesis was estimated to take 40.6 days. The primary spermatocytes life span is 13.5 days, while spermiogenesis in Piau boars lasts 14.5 days. Staging in Piau boars was based on tubular morphology system. The relative stage frequencies in these boars, based on approximately 1200 seminiferous tubule cross-sections for each animal, were as follows: stage 1, 11.7 +/- 0.7%; stage 2, 14.3 +/- 0.3%; stage 3, 5.4 +/- 0.1%; stage 4, 12.1 +/- 0.6%; stage 5, 9.6 +/- 0.4%; stage 6, 17.2 +/- 0.4%; stage 7, 15.4 +/- 0.8%; and stage 8, 14.3 +/- 0.9%. The duration of spermatogenic events and the relative stage frequencies in Piau boars differ slightly from those observed in improved swine breeds.     

Changes in the distribution of intermediate filaments in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development

BACKGROUND: Intermediate filaments (IFs) are components of the cytoskeleton. In mammalian Sertoli cell, IFs are formed by vimentin. Previous studies have shown some characteristics of its distribution in Sertoli cells, however, very little is known of its distributional changes during the seminiferous epithelium cycle and during postnatal development. METHODS: Immunohistochemical and electron microscopic methods were used to determine the distribution of vimentin-type IFs in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development. RESULTS: The distribution of IFs in adult rat Sertoli cell showed distinct cyclic changes during the seminiferous epithelium cycle. At stages I-VI, bundles of IFs extend from the perinuclear region to the supranuclear and apical regions of the Sertoli cell. These apical extensions became shorter at stage VII, and at stages VIII-X IFs were observed only in the perinuclear region. Short apical extensions reappeared at stages XI-XII; and at stages XIII-XIV, they extended again into the apical region. During this cycle, IFs were always closely associated with the heads of elongate spermatids. IFs were also shown to be in close apposition to some specialized structures on the cell membrane, such as the ectoplasmic specialization between adjacent Sertoli cells. During postnatal (p.n.) development, IFs were mainly observed at the basal nuclear region on p.n. day 7. The IFs in the supranuclear or apical regions first appeared at p.n. day 14 and gradually increased during the development. The perinuclear IFs network was fully established by p.n. day 28 and the adult distribution pattern of the IFs was established by p.n. day 42. CONCLUSIONS: Vimentin-type IFs in rat Sertoli cells are a delicate endocellular network, which is centered in the perinuclear region and extends to the apical region of the cell. During the seminiferous epithelium cycle, the distribution of IFs changes in a stage-dependent manner and is closely related to the location of the heads of elongate spermatids. During postnatal development, IFs gradually increase in numbers and the main distribution area is transferred from the basal nuclear to the perinuclear and supranuclear regions.     

Reinitiation of spermatogonial mitotic differentiation in inactive old BDF1 mouse seminiferous tubules transplanted to W/Wv mouse testis

The seminiferous epithelia of old mice (33 mo of age) are composed of spermatogonia and Sertoli cells. Histochemical examination using the anti-c-kit monoclonal antibody demonstrated that the number of differentiating type A spermatogonia decreases with age. To elucidate the differential activity of old mouse spermatogonia, we transplanted extremely thin seminiferous epithelia of old BDF, mice into W/Wv mouse testes and examined whether or not they could reinitiate differentiation. Artificially cryptorchid mice were used as the control. At 2 wk after transplantation, spermatocytes and round spermatids were detected in transplanted seminiferous tubules of the control, whereas the most advanced spermatogenic cells in those of old mice were spermatocytes. At 4 wk after transplantation, although elongated spermatids were detected in transplanted tubules of the control, haploid cells (spermatids) were still undetectable in those derived from old mice. Thus, meiosis was never restored, although spermatogonia of old mice can reinitiate differentiation into spermatocytes under suitable testicular conditions. Since it has been reported in several mammalian species that age-related changes in the testicular microenvironment lead to the gerontal cessation of spermatogenesis, the present results suggest that both a defective extratubular environment and a defective intratubular environment may cause the cessation of spermatogenesis in old BDF, mice.     

Predicting postlaryngectomy voice outcome in an era of primary tracheoesophageal fistulization: a retrospective evaluation

The aim of this study was to investigate the effect of the absence of elongate spermatids (ES) from the rat seminiferous epithelium on the quantitative secretion and synthesis of the three major Sertoli cell secretory proteins--SGP-1, SGP-2 and CP-2. Seminiferous tubules (ST) were isolated (a) from normal 28-day-old rats, in which the most mature germ cell type is the round spermatid, (b) from normal adult rats at stages IX-XIV of the spermatogenic cycle, i.e. after spermiation, or at stages I-V and VI-VIII, when ES are still attached to the Sertoli cell, and (c) at stages VI-VIII from normal adult rats and from rats treated with methoxyacetic acid (MAA) in order to specifically deplete ES at these stages. Two-dimensional SDS PAGE combined with computerized image analysis was used to analyse 35S-methionine-labelled intracellular and secreted proteins. In the case of SGP-1 and SGP-2, almost all of the protein synthesized by ST was secreted. The total amount of both SGP-1 and CP-2 secreted by unstaged ST from immature rats was significantly lower than that secreted by unstaged ST from adult rats. The total amount of SGP-1 and CP-2 secreted by adult ST at stages IX-XIV of the spermatogenic cycle also declined dramatically compared to ST at earlier stages. The proportion of the total CP-2 synthesized by ST which was secreted also declined in all situations in which ES were absent from the seminiferous epithelium. The synthesis of only SGP-2 was changed by ES depletion from ST at stages VI-VIII, which was almost doubled compared to synthesis of this protein by ST from control rats. Our results suggest strongly that the secretion of SGP-1 and SGP-2 is via the constitutive pathway, and that regulation of these two proteins by ES is at the level of protein synthesis. In contrast, the regulation of CP-2 by ES is predominantly at the level of secretion, suggesting that this protein is secreted via a regulated pathway. Our findings add to the evidence showing that ES play a major role in the regulation of Sertoli cell function.     

LDL subclass phenotypes and the insulin resistance syndrome in women

An experiment was conducted to examine the appearance of the seminiferous tubule 20 days after a single exposure of the testes of rams to a scrotal temperature of about 42 degrees C for 45 min. Ten of the animals were surgically hypophysectomized and five were simultaneously heated; these rams were treated twice a day with ovine pituitary extract to avoid modifications in the negative feedback from the testes to the pituitary and consequent changes in gonadotrophin secretion. Six intact rams (three heated and three unheated) were also studied. The pituitary extract significantly increased the testis weight and spermatogonial multiplications from A1 spermatogonia onwards. Twenty days after the heat treatment, testis weight was significantly reduced by heating; both tubular and intertubular tissues were affected. The total length of seminiferous tubules per testis was not modified, whereas the mean seminiferous tubule diameter was significantly reduced after heating. The total number of Sertoli cells per testis was not significantly modified, while their mean cross-sectional nuclear area was significantly reduced by heat treatment. A decrease in the number of all germ cells except A0 spermatogonia, from A1 spermatogonia onwards, was observed. The number of round spermatids decreased by 95 and 90%, slightly more than the diplotene primary spermatocytes (76 and 77%) and elongated spermatids (79 and 85%) in hypophysectomized pituitary extract-treated and intact rams, respectively. Round and elongated spermatids would be derived from germ cells that were respectively leptotene and young pachytene primary spermatocytes at the time of heating, whereas diplotene primary spermatocytes would have been type B spermatogonia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light cells within the limiting membrane of rat seminiferous tubules

Light-staining cells, distinct from myoid cells, were identified in electron micrographs of the limiting membrane of rat seminiferous tubules. While these cells were also found free in the interstitial space, they were observed mostly in the myoid cell layer of the limiting membrane but were never seen within the seminiferous epithelium itself. The light cells were characterized by a pale-stained cytoplasm containing a spheroidal Golgi apparatus next to a polymorphous often kidney-shaped nucleus, a few cisternae of rough endoplasmic reticulum and some granules of various types including a multivesicular bodies. In hematoxylin-stained whole mounts of dissected tubules, these light cells were readily identified under the light microscope by nuclear morphology and light-staining juxtanuclear Golgi apparatus. The incidence of these cells, per unit surface area of tubular wall, was calculated, taking into consideration the stages of the cylce of the seminiferous epithelium with which they were associated. Distributed along the entire length of seminiferous tubules, their number varied significantly during the cycle. Low numbers were found in stages II-IV and XIII of the cycle while high numbers were found in stages IX to XII and XIV-I of the cycle. These observations indicate that the seminiferous epithelium may exert an influence on the population of light cells present in the tubular limiting membrane.     

Aberrant expression of cyclin A and cyclin B1 proteins in oral carcinoma

Cyclins play an important role in regulating the passage of dividing cells through critical checkpoints in the cell cycle. Aberrant expression of cyclin proteins has been found in a number of human cancers, including carcinomas of the head and neck, where amplification of the cyclin D1 gene is a common finding. The objective of this study was to examine cell cycle kinetics in oral carcinomas by determining the expression of the S phase protein cyclin A and the M phase protein cyclin B1. Routinely processed tissue sections of 50 oral squamous cell carcinomas from the floor of the mouth were stained by immunohistochemistry for cyclin A, cyclin B1 and Ki-67 proteins. Ten specimens of normal epithelium from the floor of the mouth were used as controls. The number of cells showing nuclear staining for cyclin A, cyclin B1 and Ki-67 proteins was determined by computer image analysis. There were 17 well-differentiated, 25 moderately differentiated and 8 poorly differentiated tumours. Mean counts for cyclin A (29.50+/-4.10, mean+/-95% CI), cyclin B1 (2.05+/-0.30) and Ki-67 (49.46+/-5.91) proteins in the carcinomas were significantly higher than counts for the normal epithelial controls (cyclin A: 9.30+/-1.72; cyclin B1: 1.01+/-0.36; Ki-67: 17.40+/-4.17). For cyclin A, cyclin B1 and Ki-67, mean staining scores for all tumour grades were significantly higher than controls. There was a strong correlation between Ki-67 and cyclin A scores in all tumour groups (r2=0.68); however, the correlations between cyclin B1 and cyclin A scores (r2=0.35) and between cyclin B1 and Ki-67 scores (r2= 0.39) were weak. We conclude that there is overexpression of cyclin A and cyclin B1 proteins in oral carcinoma. Furthermore, the poor correlations for cyclin B1 scores with other cell cycle indices suggest that there may be aberrant cell cycle progression at the G2/M checkpoint in oral carcinomas.     

Three-dimensional structure of O-acetylserine sulfhydrylase from Salmonella typhimurium

The dependence of hormonal responses to exercise on sexual maturation was tested in three-year longitudinal experiment on 34 girls (11-12 years old at the beginning of the study). Sexual maturation of the girls was evaluated using Tanner scale. Girls were divided into three groups: maturation stages 1-2, 2-4 and 4-5. Children performed a 20-min cycle exercise at 60% of maximal oxygen uptake (VO max) once a year. Cortisol, insulin, somatotropin, beta-estradiol, progesterone and testosterone were determined in venous blood by RIA procedures. High basal levels of beta-estradiol and somatotropin appeared in stages 2-4 (387 +/- 92 pmol.l-1) and 12.9 +/- 2.85 ng.ml-1, respectively) and 4-5 (358 +/- 54 pmol.l-1) and 14.3 +/- 1.53 ng.ml-1, respectively). The basal progesterone level increased with maturation, testosterone appeared in the blood in stages 2-4 and 4-5. The exercise resulted in increased levels of cortisol and somatotropin, and a drop in insulin in all girls. The cortisol response was most pronounced in stage 1-2. Postexercise insulin concentration was the highest in stage 4-5. beta-estradiol level increased by 23% in stages 1-2 and 4-5, while the response was insignificant in stages 2-4. Exercise-induced progesterone increase was significant in stage 4-5. In conclusion, sexual maturation associates with several quantitative changes in exercise-induced hormonal responses.     

Characterization of the genes for the biosynthesis of the compatible solute ectoine in the moderately halophilic bacterium Halomonas elongata DSM 3043

Spontaneous germ cell death during spermatogenesis is an important event, and the usefulness of the seminiferous epithelium as an in vivo model to study apoptosis has been evidenced. Nevertheless, the response of the testis to apoptogenic agents has not been analyzed. This study was designed to determine germ cell sensitivity to induction of apoptosis and to provide baseline data on the testis response to several apoptogenic agents. Induced apoptosis was assessed by in situ DNA 3'-end labeling and quantified at every stage of the spermatogenic cycle. The shortest response time for every agent was established based on morphological and quantitative criteria. Our results show significantly increased incidence of germ cell deaths after all treatments, mainly at stages I, XII, and XIV. These specific stages coincide with those at which the greatest numbers of spontaneous germ cell deaths occur in control animals. Moreover, the rapid and highly specific response of germ cells to all the apoptogenic agents used in the present study indicate that apoptosis must be tightly regulated at these stages of the seminiferous epithelium. As a consequence, we propose that the disruption of apoptosis control might be an important determinant for idiopathic male infertility.     

Histopathology of the seminiferous tubules in mice injected with syngeneic testicular germ cells alone

Previously, a model of murine experimental autoimmune orchitis was produced by active immunization with viable syngeneic testicular germ cells without resorting to any adjuvants. The histological mode of the spermatogenic disturbance of this autoimmunity was investigated in A/J mice. A significant spermatogenic disturbance was consistently induced after the appearance of inflammatory cell responses around the tubuli recti. It first appeared seminiferous epithelium adjacent to the tubuli recti, then spread to the peripheral epithelium. The histopathology of the seminiferous tubules in the early phase ranged from partial degeneration and depletion of all kinds of germ cells to complete loss of germ cells other than some remaining spermatogonia, while both Sertoli cells and the basal lamina of the tubules appeared intact. In the late phase, depletion of Sertoli cells, disorganization of the seminiferous tubular wall or filling with many round-shaped degenerating germ cells, appearance of malformed spermatids with signet ring nuclei, depletion of immature germ cells with remaining elongated spermatids, or complete loss of the seminiferous epithelium were observed in addition to the early histopathological features.     

Development of germ cell transplants in mice

Development of spermatogonial transplants was studied by using 5- to 6-wk-old histocompatible mice as cell donors and sterile (W-locus) mice as recipients. Groups of animals transplanted with germ cell suspensions were killed at 10 min, 9 h, 24 h, 1 wk, 1 mo, 2 mo, and 3 mo along with age-matched "start" and "end" W-locus controls. Weight of testes increased significantly at 24 h through 3 mo after germ cell transplantation, suggesting that the infused cells quickly stimulated organ function. Small clones of young spermatocytes were evident at 1 mo and sperm at 2 mo. The percentage of tubular profiles containing active spermatogenesis originating from spermatogonia increased with time (0.8% at 1 mo, 8.9% at 2 mo, and 28.2% at 3 mo). Most transplanted germ cells were eliminated from the seminiferous epithelium through phagocytosis by Sertoli cells that occurred primarily before 1 wk, although some pachytene cells were able to proceed through meiosis by 1 wk. A variety of abnormal features are described that characterize developing spermatogenesis in the transplanted testis. Spermatogenesis improved quantitatively and qualitatively with time although released sperm were frequently engulfed by intratubular macrophages and Sertoli cells. A quantitative analysis of spermatogenesis from transplanted germ cells will serve as a basis for improving spermatogonial transplant efficiency.     

Effects of activin-A, inhibin-A, and transforming growth factor-beta 1 on stage-specific deoxyribonucleic acid synthesis during rat seminiferous epithelial cycle

Activin and inhibin are members of the transforming growth factor-beta (TGF beta) gene family. They are expressed in various organ systems, where they possess regulatory functions. Inhibin, activin, and TGF beta have been reported to also be expressed in the adult rat testis. We studied in vitro the action of these growth factors on premitotic and premeiotic DNA synthesis during the rat seminiferous epithelial cycle. Two-millimeter rat seminiferous tubule segments were isolated by transillumination-assisted microdissection from stages V, VIIa, VIII-IX, and I of the cycle and incubated in vitro in the presence of activin-A, inhibin-A, or TGF beta 1. During 24-, 48-, and 72-h incubation spontaneous progression of spermatogenesis was noted. The staged samples allowed us to selectively quantitate DNA synthetic activity of specific germ cell types. At the end of the culture, the tubules were pulse labeled with [3H]thymidine, and DNA synthesis was quantified by liquid scintillation counting, and the activated cells were detected by autoradiography. Activin-A stimulated preleptotene spermatocyte DNA synthesis in a dose-dependent manner. DNA synthesis of intermediate spermatogonia was also stimulated by activin-A, whereas inhibin-A inhibited DNA synthesis of these cells. TGF beta 1 had a small, but significant, stimulatory effect on DNA synthetic activity at stage VII. These results support the view that activin-A, inhibin-A, and TGF beta 1 take part in the regulation of DNA synthesis during rat spermatogenesis.     

Cell cycle regulation and differentiation in the human podocyte lineage

Mature podocytes are regarded as growth-arrested cells with characteristic phenotypic features that underlie their function. To determine the relationship between cell cycle regulation and differentiation, the spatiotemporal expression of cyclin A, cyclin B1, cyclin D1, the cyclin-dependent kinase inhibitors (CKIs) p27 and p57, and markers of differentiating podocytes in developing human kidneys was investigated by immunohistochemistry. In S-shaped body stage, Ki-67, a cell proliferation marker that labels the G1/S/G2/M phase, was expressed in the majority (more than 80%) of presumptive podocytes, along with cyclin A (approximately 20% of the Ki-67-positive cells) and cyclin B1 (less than 5% of Ki-67-positive cells) expression. Among these cells), cyclin D1 and CKIs were markedly down-regulated. At the capillary-loop stage, by contrast, CKIs and cyclin D1 were intensely positive in podocytes, whereas no Ki-67, cyclin B1, or cyclin A expression was seen. Moreover, double-immunolabeling and serial-section analysis provided evidence that CKIs and markers specific for differentiating podocytes, namely PHM-5 (podocalyxin-like protein in humans), synaptopodin (a foot process-related protein), and C3b receptor, were co-expressed at the capillary-loop stage. Podocytes were the only cells within the glomeruli that expressed CKIs at immunohistochemically detectable levels. Furthermore, bcl-2 (an apoptosis inhibitory protein) showed a reciprocal expression pattern to that of CKI. These results suggest that 1) the cell cycle of podocytes is regulated by cyclin and CKIs, 2) CKIs may act to arrest the cell cycle in podocytes at the capillary-loop stage, and 3) the specific cell cycle system in podocytes may be closely correlated with their terminal differentiation in humans.